Myosin and the enzyme, myosin light chain kinase, in primary cultures of smooth muscle cells derived from rat aorta have been studied in comparison with those in intact aorta. Postconfluent primary cultures were found to contain three different myosin heavy chains (MHCs) following SDS-polyacrylamide gel electrophores (SDS-PAGE). Based on their migration in SDS-PAGE, antigenicities, and 2-dimensional peptide maps of iodinated MHCs, the two slowest migrating MHCs have similar properties to those found in intact smooth muscle tissues, including rat aorta. The fastest migrating MHC was distinct from smooth muscle myosins but was very similar to nonmuscle myosin prepared from platelets or fibroblasts. At the subconfluent stage, the greater part of MHCs was nonmuscle myosin. These results suggest that smooth muscle cells contain predominantly nonmuscle myosin while actively growing, but at a post confluent stage, contain a mixture of both smooth muscle and nonmuscle myosins. Presently, studies are under way to determine whether a single cell in postconfluent primary culture contain both smooth muscle and nonmuscle myosins. The protein structures of two MHCs of smooth muscle is also being investigated. Using antibodies to turkey gizzard myosin light chain kinase, two proteins having Mr of 130,000 and 85,000 were recognized on immunoblots prepared from primary culture of rat aorta smooth muscle cells. The 130,000-dalton peptide was also found in the intact aorta. On the other hand, the 85,000-dalton peptide was not detected in intact aorta but was found in nonmuscle cells as well as late passage subcultures of smooth muscle cells. The question of whether the 85,000-dalton peptide is a unique species of myosin kinase or is simply a proteolytic product of the 130,000-dalton enzyme has been raised. The enzymatic properties and protein structure of the 85,000-dalton peptide is under study.